Environmental sampling articles and methods

ABSTRACT

The present invention refers to articles for collecting samples from a surface, articles for microbiological analyses of said samples, and methods of use of said articles. The articles include sample collectors, sample housings with optional barrier layers, and sample-ready reagent strips comprising hydrophilic agents to grow and detect microorganisms. The disclosure includes methods to collect, detect, and quantify microorganisms in a surface sample.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Patent Application No.60/989,356, filed on Nov. 20, 2007 and which is incorporated herein byreference in its entirety.

BACKGROUND

When surfaces become contaminated with bacteria, fungi, yeasts, viruses,or other microorganisms, or “microbes,” sickness (morbidity) and,sometimes, death (mortality) may result. This is particularly true whensurfaces in food processing plants and healthcare facilitates (e.g.,hospitals) become contaminated with microorganisms.

In food processing plants, surfaces (e.g., solid surfaces, equipmentsurfaces, protective clothing, etc.) may become contaminated. Suchcontamination may be caused by or transferred to meat or other foods. Inhealthcare facilities, microbes may be released onto surfaces (e.g.,solid surfaces, equipment surfaces, clothing, etc.) from infectedindividuals or otherwise. Once a surface becomes contaminated withmicrobes, contact with the contaminated surface may easily and readilytransfer microbes to other locations, such as another surface, anindividual, equipment, food, or the like.

As is well known, microbial contamination and transfer in certainenvironments may pose significant health risks. For example, the foodthat leaves a contaminated food processing plant will subsequently beeaten, and may cause sickness and, possibly, death. Microorganisms suchas Listeria monocytogenes, Salmonella enteriditis, and Escherichia coliO157:H7 are of particular concern.

Microbial contamination is of concern in healthcare facilities sincesome of the patients of such facilities often suffer from infections bypathogenic microbes and, thus, bring the pathogenic microbes into suchfacilities. Further, many of those who are present in such facilities(e.g., patients) are sick and may be immunologically compromised. Theseindividuals are, thus, at increased risk of becoming sick from infectionby the contaminating microbes.

In view of the potential dangers of microbial contamination, inparticular the ease with which microbes may be transferred in certainenvironments and the health hazards associated with the contamination ofcertain environments, a variety of techniques have been developed andemployed to detect such contamination so that it may be promptlyremedied.

Conventionally, environmental microbial testing includes obtaining asample from a surface. This is typically done by contacting (e.g.,wiping, swiping, etc.) the surface with a sterile sampling appliance,such as a swab or a sponge. Surfaces that are tested in this manner areusually quite clean; thus, the number of microorganisms that are pickedup by the sampling appliance is typically quite low. Due to the smallnumber of microorganisms, any microbes that are on (e.g., picked up by)the sampling appliance typically must be reproduced, or “grown” or“cultured,” to provide a sufficient number of organisms for furtheranalysis. Accordingly, at least a portion of the sample is thentypically neutralized and, optionally, stabilized, repaired, orenriched, then applied (e.g., transferring, swiping, dipping andagitating, etc.) to an appropriate growth media (e.g., agar (a gelatinor gelatin-like material), broth (a liquid), etc.), which includesnutrients that will help microbes of interest grow. The growth media maybe selective, meaning that the growth media may include ingredients thatwill allow some microorganisms to grow at faster rates than othermicrobes or it may include ingredients that will prevent the growth ofat least some undesired microbes. The growth media is incubated or heldat a certain temperature for a predetermined period of time, typicallyabout 24 to about 48 hours, or until microbial growth is visiblyapparent.

Once the sample has had a sufficient opportunity to grow, the amount ofbacteria (e.g., the number of colonies on an agar plate) that has grownmay then be evaluated (e.g., by an individual or with automatedequipment) to provide some indication of the number and type of microbesthat were present on a certain area of the surface at the time thesample was taken-usually a day or two earlier. Immunological or othertesting may also be performed to determine or confirm the identity oridentities of any microbes of interest that were present in the sample.

For example, when testing for a Salmonella species of bacteria, a samplepotentially including the Salmonella species may be applied to aselective growth media. The selective growth media may then be incubatedfor a period of about 24 to about 48 hours until growth of Salmonellamicrobes is visible. Once Salmonella colonies are visibly present on theselective growth media, the colonies may be evaluated to confirm theiridentities, and, optionally, counted to estimate a number of Salmonellamicroorganisms present on a certain area of the tested surface.Alternatively or additionally, the cultured microorganisms may besubjected to an immunoassay or nucleic acid assay to more directlyconfirm their identities.

Simpler, rapid, accurate tests for environmental organisms are needed.This invention provides devices and methods for such tests.

SUMMARY

In one aspect, the invention includes an article for sampling anddetecting surface microorganisms. The article can include a samplecollector substantially free of hydrophilic agents and a reagent stripattached thereto. The sample collector can include a substrate withupper and lower major surfaces wherein at least one major surface iswater-impervious and wherein at least one major surface comprises aporous material. The reagent strip can include a self-supportingsubstrate with upper and lower major surfaces wherein at least part ofone major surface is coated with a cold water soluble gelling agent anda hydrophilic agent selected from the group consisting of a nutrient forgrowing microorganisms, a selective agent, a buffer, an indicator, andcombinations of two or more of the foregoing. Optionally, the articlemay further include a barrier layer.

In another aspect, the invention includes an article for detecting orenumerating microorganisms. The article can include a bottom member anda cover sheet attached thereto. The bottom member can include aself-supporting water impervious substrate with upper and lower majorsurfaces wherein the upper major surface comprises an attachmentstructure. The cover sheet can include upper and lower major surfaceswherein at least part of the lower major surface of the cover sheet iscoated with a cold water soluble powder including at least one gellingagent. The upper major surface of the bottom member faces the lowermajor surface of the cover sheet

In another aspect, the invention includes a sample collector forcollecting environmental surface samples. The sample collector caninclude a substrate with upper and lower major surfaces wherein at leastone major surface is water impervious, wherein at least one majorsurface comprises a porous material, and wherein the sample collector issubstantially free of hydrophilic agents.

In another aspect, the invention includes a sample housing comprising abottom member, a spacer, a cover sheet, and a water-resistant barrierlayer. The bottom member can include a self-supporting water-impervioussubstrate with upper and lower major surfaces. The spacer can include anaperture and is adhered to the upper surface of the bottom member. Thecover sheet can include a substrate with upper and lower major surfaceswherein at least part of one major surface is coated with a cold watersoluble powder including at least one gelling agent. The water-resistantbarrier layer is positioned between the cover sheet and the spacerwhereby a coated surface of the cover sheet faces the barrier layer.

In another aspect, the invention includes a reagent strip for thedetection of microorganisms. The reagent strip includes aself-supporting substrate with upper and lower major surfaces and a drycoating on at least a part of both major surfaces. The coating caninclude a hydrophilic agent comprising a nutrient for growingmicroorganisms and an indicator and a cold water soluble gelling agent.

In another aspect, the invention includes a kit for sampling anddetecting surface microorganisms. The kit can include a sample collectorand a reagent strip. The sample collector is substantially free ofhydrophilic agents and can include a water-impervious substrate withupper and lower major surfaces wherein at least one major surfacecomprises a porous material. The reagent strip can include aself-supporting substrate with upper and lower major surfaces wherein atleast part of one major surface is coated with a cold water solublegelling agent and a hydrophilic agent selected from the group consistingof a nutrient for growing microorganisms, a selective agent, a buffer,an indicator, and combinations of any two or more of the foregoing.

In another aspect, the invention includes a kit for sampling andenumerating microorganisms. The kit can include a sample collector, asample housing and, optionally, a reagent strip. The sample collectorcan include a substrate with upper and lower major surfaces wherein atleast one major surface is water-impervious. The sample housing caninclude a bottom member and a cover sheet. The bottom member can includea self-supporting water impervious substrate with upper and lower majorsurfaces. The cover sheet can include a substrate with upper and lowermajor surfaces wherein at least part of one major surface is coated witha cold water soluble powder including at least one gelling agent.

In another aspect, the invention includes a kit for sampling andenumerating microorganisms. The kit can include a sample collector, acover sheet, and a bottom member. The sample collector consistsessentially of a substrate with upper and lower major surfaces whereinat least one major surface is water-impervious. The cover sheet caninclude a substrate with upper surface and lower major surfaces whereinat least part of one major surface is coated with a cold water solublepowder including at least one gelling agent. The bottom member caninclude a self-supporting water-impervious substrate with upper andlower surfaces and an attachment structure.

In another aspect, the invention includes a method for detectingmicroorganisms on an environmental surface. The method can includeproviding a liquid sample-suspending medium, sample collector, a samplehousing, and a reagent strip. The sample collector can include asubstrate with upper and lower major surfaces wherein at least one majorsurface is water-impervious. The sample housing can include a bottommember and a cover sheet comprising a substrate with upper and lowermajor surfaces wherein at least part of one major surface of the coversheet is coated with a cold water soluble powder including at least onegelling agent. The reagent strip can include a self-supporting substratewith upper and lower major surfaces wherein at least part of one majorsurface is coated with a cold water soluble gelling agent and ahydrophilic agent selected from the group consisting of a nutrient forgrowing microorganisms, a selective agent, a buffer, an indicator, andcombinations of any two or more of the foregoing. The method further caninclude obtaining a sample on at least one major surface of the samplecollector, forming an assembly by placing the sample-collector into thesample housing with the at least one major surface comprising the sampleoriented toward the cover sheet, applying the sample-suspending mediumto the sample collector major surface comprising the sample, contactingthe lower surface of the cover sheet with the sample-suspending mediumto form a hydrated gel, placing a coated surface of the reagent strip incontact with the hydrated gel, incubating the assembly for a period oftime, and observing an indicator of microbial growth

In another aspect, the invention includes a method for detectingmicroorganisms on an environmental surface. The method can includeproviding barrier layer, a sample-suspending medium, a sample collector,a sample housing, and a reagent strip. The sample collector can includea substrate with upper and lower major surfaces wherein at least onemajor surface is water-impervious. The sample housing can include abottom member comprising a self-supporting water impervious substratewith upper and lower major surfaces and a cover sheet comprising asubstrate with upper and lower major surfaces wherein at least part ofone major surface is coated with a cold water soluble powder includingat least one gelling agent. The method further can include obtaining asample on at least one major surface of the sample collector, forming anassembly by placing the sample-collector into the sample housing withthe at least one major surface comprising the sample oriented toward thecover sheet, applying the sample-suspending medium to the samplecollector major surface comprising the sample, incubating the samplehousing for a period of time, removing at least a portion of the barrierlayer from the sample housing, contacting the lower surface of the coversheet with the sample-suspending medium to form a hydrated gel, placinga coated surface of the reagent strip in contact with the hydrated gel,incubating the assembly for a period of time, and observing an indicatorof microbial growth.

In another aspect, the invention provides a method of detecting Listeriaspecies in an environmental sample. The method can include providing asample collector with a surface, a sample suspending medium and areagent strip; obtaining a sample on the surface of the samplecollector; hydrating the sample or the reagent strip; and contacting thereagent strip and the sample. The reagent strip includes a dry coatingthat can include a hydrophilic agent comprising a nutrient for growingmicroorganisms, an indicator; and a cold water soluble gelling agent

In another aspect, the invention provides a method of detecting Listeriaspecies in an environmental sample. The method can include providing asample collector with a surface, a sample suspending medium, a samplehousing, and a reagent strip; obtaining a sample on the surface of thesample collector; placing the sample collector into the sample housing;hydrating the sample or the reagent strip; and contacting the reagentstrip and the sample. The reagent strip can include a dry coating thatincludes a hydrophilic agent comprising a nutrient for growingmicroorganisms, an indicator; and a cold water soluble gelling agent.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a sample housing that comprises“a” barrier layer can be interpreted to mean that the sample housing caninclude “one or more” barrier layers.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further explained with reference to the drawingfigures listed below, where like structure is referenced by likenumerals throughout the several views.

FIG. 1 is a perspective view of an exemplary embodiment of a samplecollector according to the present invention.

FIG. 2A is a top perspective view of an exemplary embodiment of atextured sample collector according to the present invention.

FIG. 2B is a perspective view of an exemplary embodiment of a samplecollector comprising a porous material according to the presentinvention.

FIG. 3A is a cross-sectional schematic view of an exemplary embodimentof a sample housing comprising a spacer according to the presentinvention.

FIG. 3B is a plan view of the sample housing of FIG. 3A.

FIG. 4 is a cross-sectional schematic view of one embodiment of anarticle for sample collection and enumeration of microorganisms.

FIG. 5 is a perspective schematic view of one embodiment of a reagentstrip according to the present invention.

FIG. 6 is a cross-sectional schematic view of an alternative embodimentof an article for sample collection and enumeration of microorganisms.

FIG. 7 is a cross-sectional schematic view of an alternative embodimentof an article for sample collection and enumeration of microorganisms.

FIG. 8 is a cross-sectional schematic view of an alternative embodimentof an article for sample collection and enumeration of microorganisms.

DETAILED DESCRIPTION

The present disclosure provides articles and methods for samplingsurfaces, temporarily storing the sample, and growing and detectingmicroorganisms present in the sample. As discussed in detail below, theinventive devices include the individual elements of a sample collector,a sample housing, a reagent strip, a barrier layer, combinations of twoor more of the foregoing elements, and methods of using such devices.The present disclosure also includes kits for sampling surfaces anddetecting microorganisms and methods of using such kits. Sometraditional surface microbial sampling procedures involve an enrichmentculture procedure, after which a fraction of the enrichment culture istested for target microorganisms using subsequent biochemical,immunological, or genetic test procedures. Other traditional surfacemicrobial sampling procedures use a moistened sponge or swab to wipe thesurface. The sponge is subsequently extracted with neutralizing bufferor growth media and the portion of the target microorganisms that can beextracted from the sponge or swab is tested for the presence of targetmicroorganisms. In contrast, the devices and methods of the presentdisclosure provide means to test the entire original sample collectorand, thereby, are more likely to detect the presence of very low numbersof microorganisms on a surface. Furthermore, the devices and methods ofthe present disclosure are simpler and more convenient than traditionalmethods for detecting organisms in a surface sample.

Devices of the present disclosure provide a marked improvement overprior art devices and techniques which rely on standard agar platingmethods as well as other microbiological testing. The media coated onthe devices of the present invention do not contain matrixes which wouldadversely affect one's ability to visualize and isolate bacterialcolonies. Not only will the media provided by the methods and devicesallow enumeration of the bacterial colonies growing in the devices, butthe colonies may be easily isolated for further testing in the samemanner as bacterial colonies growing on conventional agar medium in apetri dish. The devices have the added feature of being much morecompact and light-weight than petri dishes and take up less space in thelaboratory. Furthermore, the devices are completely disposable allowingfor safer and more rapid clean-up after use.

Sample Collector

FIG. 1 illustrates one embodiment of a sample collector 110 having arelatively smooth substrate 112 for sampling an environmental surface orpatient surface. The substrate 112 has an upper major surface 114 and alower major surface 116. At least one of the major surfaces of thesubstrate 112 is water impervious and/or may comprise a water-imperviouscoating. In some embodiments, the sample collector 110 is relativelyflexible, allowing it to conform to and maintain contact with unevensurfaces. At least one of the major surfaces of the sample collector 110is brought into contact with the sample area to effect transfer ofmaterial, such as liquids, solids, semi-solids, or combinations thereof,from the sample area to the sample collector 110. The sample collector110 may be used to collect material from a sample area, such as a foodcontact surface or a superficial wound, which is to be analyzed.

The substrate 112 of the sample collector 110 may be constructed fromvarious materials. Nonlimiting examples of suitable materials forinclude plastic films, such as polyethylene, polypropylene, orpolyester; cellulosic materials, such as paper or cardboard, whichcomprise a coating or a composition to render at least one major surfacewater-impervious; foams, such as polyethylene or polystyrene foam; orfabrics which have at least one major surface that is water-impervious.

In some embodiments, at least one major surface of the sample collector110 is textured. As used herein, the word “textured” refers to theprofile of the surface that is used to collect the sample material. Forexample, the texture of the major surfaces of the sample collector 110may be relatively smooth, as illustrated in FIG. 1. Alternatively, thetexture of the major surfaces of the sample collector 110 may berelatively rough. FIG. 2A illustrates one embodiment of a samplecollector 210 including a major surface 214 with a relatively roughtexture. In this embodiment, the substrate 212 may comprise acombination of raised structures 218 and recessed structures 220.Alternatively, the substrate 212 may comprise raised structures 218 orrecessed structures 220. Raised structures 218 may be present in varioussizes and may be present in various shapes, such as bumps, spikes,ridges, and the like, or combinations thereof. The raised structures 218may be randomly distributed across the surface of the substrate 212 or,alternatively, may be uniformly spaced apart. Recessed structures 220may be present in various sizes and may be present in various shapes,such as holes, pits, valleys, troughs, channels, microchannels, and thelike, or combinations thereof. The raised structures 218 or recessedstructures 220 may be an integral part of the substrate 212 materialfrom which the sample collector 210 is made. Alternatively, thestructures may be bonded to the substrate 212. Without being bound bytheory, it is believed that a sample collector 210 comprising eitherraised structures 218 or recessed structures 220 may provide anadvantage in collecting sample material from an environmental surface ora patient surface by providing structures that can trap sample materialand/or abrade and collect material from the surface to be sampled. Alsoillustrated in FIG. 2A is an optional tab 228, which provides aconvenient area to hold the sample collector 210 while collecting thesample.

FIG. 2B illustrates a sample collector 210 with an alternative texturedsurface. The sample collector 210 can comprise a bonded material 224,such as a nonwoven, adhered to a major surface of the substrate 212 bymeans of an adhesive layer 222. Adhesive layer 222 may bewater-insoluble, should be non-inhibitory to the growth ofmicroorganisms, and should be capable of withstanding the sterilizationprocess. Preferably, the adhesive layer 222 and the bonded material 224are sufficiently transparent when wet to enable the viewing of bacterialcolonies through the substrate 212 coated with the adhesive layer 222.In some embodiments, the adhesive layer 222 includes apressure-sensitive adhesive. An exemplary embodiment of a bondedmaterial 224 adhered to the substrate 212 includes a nonwoven material,such as a knitted loop nonwoven obtained from Gehring Textiles, Inc.(Garden City, N.Y.) or glass fiber filter, adhered to a plasticsubstrate 212, such as white polyester (5 mil (0.13 mm) thickness), bymeans of a pressure-sensitive adhesive layer 222, such as a tackifiedhigh pressure sensitive iso-octyl acrylate/acrylic acid copolymeradhesive (96 wt. % iso-octyl acrylate and 4 wt. % acrylic acid)Alternatively, the bonded material 224 may comprise other texturedmaterials, such as foams (e.g., polyurethane foam), fabrics, orcellulosic materials (e.g. filter paper). Also shown in FIG. 1C is a tab228, which is conveniently detachable from sample collector 210 by meansof a perforation 226.

Sample Housing

The sample housing is an article into which a sample collector, withsample material disposed thereon, may be placed and, optionally, storedfor a period of time. The sample housing further may be used as anarticle into which solutions and/or reagents may be added to facilitatethe growth, detection, or enumeration of microorganisms. In certainembodiments, the sample housing may be used as an article in which asample is incubated with a reagent strip and/or reagents to facilitatethe growth, detection or enumeration of microorganisms.

FIG. 3A illustrates an exemplary sample housing 360. The sample housing360 comprises a bottom member 350, a barrier layer 370, and a coversheet 330.

The bottom member 350 comprises a self-supporting water-impervioussubstrate 352 with upper and lower major surfaces. The bottom member 350is preferably a relatively stiff material such as polyester,polypropylene or polystyrene film, which will not absorb or otherwise beaffected by water. For example, polyester films approximately 0.004 to0.007 inch (0.1-0.2 mm) thick, polypropylene films approximately 0.004to 0.008 inch (0.1-0.2 mm) thick and polystyrene films approximately0.015 inch (0.38 mm) thick should work well. Other suitable substratesinclude paper with a polyethylene or other water-proof coating. Thebottom member 350 may be either transparent or opaque, depending onwhether one wishes to view bacterial colonies through the bottom member350. To facilitate the counting of bacterial colonies, the bottom member350 may have a square grid pattern printed thereon as described in U.S.Pat. No. 4,565,783, which is incorporated herein by reference. Thematerials used to construct the bottom member 350 should be relativelyinert to microorganisms and should be compatible with the sterilizationprocess.

In this embodiment, the bottom member 350 is further comprised of anadhesive layer 354 disposed on the upper surface of the substrate 352and a spacer 356. The adhesive layer 354 forms an attachment between thespacer 356 and the substrate 352. The adhesive layer 354 should beselected according to the guidelines for the adhesive layer 222 of thesample collector 210 described above. The spacer 356, which comprises anaperture 357, should be constructed from a water-insoluble material. Thewalls of the aperture 357 provide a well of predetermined size and shapeto confine a volume of liquid medium added to the sample housing 360.The spacer 356 should be thick enough and the aperture 357 large enoughto form a well of the desired volume, e.g., 1 milliliters, 2milliliters, 3 milliliters, or 5 milliliters. Closed cell polyethylenefoam or polystyrene foam are suitable materials for the spacer 356, butany material which is hydrophobic (non-wetting), inert tomicroorganisms, and, preferably, capable of withstanding a sterilizationprocess may be used. As illustrated in FIG. 3A, the bottom of the wellformed by the aperture 357 may comprise an adhesive layer 354, which maybe used to secure a sample collector (not shown in FIG. 3A).Alternatively, the bottom of the well formed by the aperture 357 maycomprise attachment structures (described below) or no adhesive layer354 or attachment structures.

Attached to the upper surface of the spacer 356 is a barrier layer 370.In this embodiment, the spacer 356 and the barrier layer 370 are joinedtogether by a double-sided adhesive tape 340. The barrier layer 370should be water-resistant. Barrier layer 370 is preferably transparentto permit the observation of objects located beneath the barrier layer370 and is substantially impermeable to bacteria, water and water vapor.Generally, the barrier layer 370 can have the same properties as bottommember 350, but need not be as stiff. Exemplary materials for barrierlayer 370 include, for example, polypropylene film (e.g., 1.6 milbiaxially-oriented polypropylene (BOPP)) or polyethylene film. Referringback to FIG. 3A, the barrier layer 370 is shown with an optionalperforation 372, which can be torn easily to permit the removal of atleast a part of the barrier layer 370 from the sample housing 360. Thebarrier layer 370 also is shown with an optional extension, or tab 374,which allow the barrier layer 370 to be grasped easily to tear theperforation 372.

Attached to the upper surface of the barrier layer 370 is a cover sheet330. The barrier layer 370 and the cover sheet 330 are joined togetherby a double-sided adhesive tape 340. The attachment of the spacer 356 tothe barrier layer 370 and the cover sheet 330 near an edge of the samplehousing 360 conveniently forms a hinge region 345 allowing the barrierlayer 370 and/or the cover sheet 330 to be lifted, thus exposing innerportions of the sample housing 360 while retaining the alignment of thecomponent parts. Certain low-adhesion adhesive mixtures, such as thosedescribed in U.S. Pat. No. 5,118,750, may be used on at least one sideof the double-sided adhesive tape 340 to form a detachable attachmentbetween the elements (e.g., cover sheet, barrier layer, and bottommember). The cover sheet 330 and barrier layer 370 may alternatively bejoined to the bottom member 350 by other means, for example ultrasonicwelding, clamping, or stapling.

The cover sheet 330 comprises a substrate 332 with upper and lower majorsurfaces. Coated on at least part of the lower major surface (facing thebarrier layer 370) is an adhesive layer 334. A cold water soluble powder336 including at least one gelling agent is adhered to the adhesivelayer 334. Alternatively, at least a portion of the cover sheet 330,such as the hinge region 345, may be coated with only an adhesive or maybe substantially free of any type of coating.

Adhesive layer 334 should be coated onto cover sheet 330 at a thicknesswhich is preferably less than the diameter of the particles of thepowdered gelling agent and/or nutrients. The object is to apply enoughadhesive in adhesive layer 334 to adhere the particles to the substratebut not so much that the particles become completely embedded in theadhesive. A uniform monolayer of powder 336 is desired with sufficientsurface area exposed for hydration. Generally, an adhesive layer 334 inthe thickness range of 0.0002 to 0.0005 inch (0.005-0.012 mm) issuitable. An exemplary adhesive used in adhesive layer 334 is acopolymer of isooctylacrylate/acrylamide (in a mole ratio of 94/6).Other pressure sensitive adhesives which may be used includeisooctylacrylate/acrylic acid (in a mole ratio of 95/5 or 94/6) andsilicone rubber. Adhesives which turn milky upon exposure to water areless preferred, but may be used in conjunction with a non-transparentsubstrate or where colony visualization is not required.

A monolayer of cold-water-soluble powder 336 is adhered uniformly toadhesive layer 334. Powder 336 may comprise a gelling agent or a mixtureof gelling agents. As used in the specification and claims, the term“powder” designates a finely divided particulate material having anaverage diameter of less than 400 micrometers. As used in thespecification and claims, the term “cold-water-soluble” designatesmaterial which forms a solution in water at room temperature. Suitablegelling agents for inclusion in powder 336 include both natural andsynthetic gelling agents which form solutions in water at roomtemperature. Gelling agents such as hydroxyethyl cellulose,carboxymethyl cellulose, polyacrylamide, locust bean gum and algin, formsolutions in water at room temperature and are suitable gelling agentsfor providing powders which are “cold-water-soluble.” The preferredgelling agents for powder 336 are guar gum and xanthan gum, thesegelling agents being useful individually or in combination with oneanother.

The gelling agent is preferably present in an amount sufficient to forma substantially transparent gel having a Brookfield viscosity of atleast 1500 cps. A sufficient amount of the gelling agent should beadhered to the cover sheet 330 so that a predetermined quantity of wateror an aqueous sample, e.g., 1-3 milliliters, placed in the samplehousing 360 will form a gel having a viscosity of about 1500 cps or morewhen measured at 60 rpm with a Brookfield Model LVF viscometer at 25° C.Gels of this viscosity will allow convenient handling and stacking andprovide distinct colony identification. In most cases 0.025 to 0.050gram of guar gum on a surface area of 3.14 in² (5.07 cm²) will provide asufficiently viscous gel when hydrated with 1-3 milliliters of anaqueous sample. The size of the particles of powder 336 may be used tocontrol the coating weight per unit area. For example, approximately 100mesh guar gum coats to a weight of about 0.05 grams/2-inch (5.1 cm)diameter disc; and a 400 mesh guar gum coats to a weight of about 0.025grams/2-inch (5.1 cm) diameter disc.

The cover sheet 330 provides a means for covering the sample housing 360to prevent contamination during sample storage and/or incubation. Thecover sheet 330 can be a water-resistant sheet attached, directly orindirectly, in hinge region-like fashion to one edge of the bottommember 350. Cover sheet 330 is preferably transparent to facilitatecounting of the bacterial colonies and is substantially impermeable tobacteria and water vapor. As used herein, “substantially impermeable tobacteria and moisture vapor” designates a cover sheet 330 which preventsundesired contamination of the sample housing 360 during shipping,storage and use of the devices and which provide an environment whichwill support the growth of microorganisms during the incubation period.Generally, it can have the same properties as bottom member 350, butneed not be as stiff. Cover sheet 330 can be selected to provide theamount of oxygen transmission necessary for the type of microorganismdesired to be grown. For example, polyester films have low oxygenpermeability (less than 5 g/100 in² (645 cm²)/24 hours per 0.001 inch(0.025 mm) of thickness) and would be suitable for growing anaerobicbacteria. On the other hand, polyethylene has very high oxygenpermeability (approximately 500 g/100 in² (645 cm²)/24 hours per 0.001inch (0.025 mm) of thickness) and would be suitable for aerobicorganisms. A preferred material for cover sheet 330 is a 1.6 milbiaxially-oriented polypropylene film.

The cover sheet 330 simply can be lifted, a liquid sample-suspendingmedium can be placed on the substrate, and the cover sheet 330 then canbe returned to its original position thereby sealing in the gelledmedium. The cover sheet 330 is preferably transparent to allow thebacterial colonies to be seen. The materials used to form the coversheet 330 may be conveniently selected to obtain the desiredpermeability to gases such as oxygen.

FIG. 3B shows a top view of the sample housing 360 of FIG. 3A. The hingeregion 345 is located along the top edge of the sample housing 360. Theoptional perforation 372 is adjacent to the hinge region 345. Thecircular aperture 357 in the spacer 356 is located in the central regionof the sample housing 360. The tab 374 of the barrier layer 370 can beseen extending beyond the edge of the cover sheet 330 at the end of thesample housing 360 opposite the hinge region 345.

FIG. 4 illustrates an alternative embodiment of a sample housing 460.The sample housing 460 is comprised of cover sheet 430, barrier layer470, and bottom member 450 elements, secured at a hinge region 445 by,for example, double-sided adhesive tape 440. In this embodiment, thebottom member 450 comprises a substrate 452 and an optional attachmentstructure 458. The attachment structure 458 may comprise an adhesive, acomponent of a hook-and-loop attachment means, a stem web structure suchas those described in U.S. Patent Application Publication No.2003/0088946A, or the like, which can be used to hold a sample collectorin place. The barrier layer 470 may comprise a perforation 472 and/or atab 474 and can be constructed as described above. In certainembodiments, the lower major surface of the barrier layer 470 maycomprise a release material, to prevent adhesion to the attachmentstructure 458. Release coatings are known in the art and are routinelyused to reduce the adhesion between adhesives and plastic films. Thecover sheet 430 comprises a substrate 432, adhesive layer 434, andpowder 436 and can be constructed as described above.

Reagent Strip

FIG. 5 illustrates one embodiment of a reagent strip 580 according tothe present invention. The reagent strip 580 is comprised of a substrate582 with upper and lower major surfaces. In this embodiment, at least apart of both major surfaces of the substrate 582 is coated with anoptional adhesive 584 and a hydrophilic agent 586. In some embodiments,at least a part of one major surface of the substrate 582 is coated withthe optional adhesive 584 and/or the hydrophilic agent 586. In someembodiments, the reagent strip 580 comprises an optional tab 588. Thetab may include an optional perforation 589, which allows the tab 588 tobe separated from the reagent strip 580.

The detection of microorganisms in a sample often involves the use ofreagents, such as hydrophilic agents, to promote growth, inhibit growth,and/or detect a metabolic activity of certain microorganisms.“Hydrophilic agents”, as used herein, include nutrients (e.g., proteins,peptides, carbohydrates, vitamins) at concentrations which may be usedto promote the growth of certain microorganisms, salts (e.g., NaCl,LiCl, potassium tellurite) or inhibitors (e.g. naladixic acid,aztreonam, other antibiotics, dyes) at concentrations which may be usedto selectively inhibit the growth of certain microorganisms, dyes orindicators (e.g. triphenyltetrazolium chloride, chlorophenol red,bromothymol blue, o-nitrophenylphosphate,5-bromo-4-chloro-3-indolyl-β-glucopyranoside) at noninhibitoryconcentrations which may be used to detect metabolic activity such anenzyme activity or a fermentation process, and gelling agents (e.g.,agar, xanthan gum, guar gum).

The adhesive 584 may be required when coating certain hydrophilic agents586, such as powders, onto the substrate 582, as discussed above. Insome embodiments, the hydrophilic agent 586 comprises a gelling agentwhich, when hydrated to form a gel, may be coated directly onto thesubstrate 582. The substrate 582 may be selected from a number ofmaterials, such as the materials described above for the construction ofbottom member, the barrier layer, and the cover sheet. In certainembodiments, the substrate 582 of the reagent strip 580 isfree-standing. In certain embodiments, the substrate 582 of the reagentstrip 580 may be substantially impervious to water. In alternativeembodiments, the substrate 582 of the reagent strip 580 may bewater-absorbent, such as a filter paper or a hydrophilic foam. Incertain embodiments, the reagent strip 580 may be constructed frompolyethylene-coated paper and may comprise an optional printed grid (notshown) to facilitate counting colonies of microorganisms.

It may be desirable to incorporate a dye into the hydrophilic agents 586included on the reagent strip 580. The dye may be incorporated into agel or powder mixture coated on the substrate 582. Alternatively, thedye may be incorporated into the adhesive 584. Suitable dyes are thosewhich are metabolized by the growing microorganisms, and which cause thecolonies to be colored for easier visualization. Examples of such dyesinclude triphenyltetrazolium chloride, p-tolyl tetrazolium red,tetrazolium violet, veratryl tetrazolium blue and related dyes. Othersuitable dyes are those sensitive to pH changes such as neutral red orchlorophenol red.

The material employed in the hydrophilic agent 586 on the reagent strip580 is cold-water-reconstitutable. As used herein,“cold-water-reconstitutable” designates material which forms a solution,sol or gel in water at room temperature. Suitable gelling agents forinclusion in the coating of this embodiment (if such are contained inthe coating) include the above-described gelling agents, such as guargum and locust bean gum, which form solutions in water at roomtemperatures.

The reagent strip may be used to select for the growth of certainorganisms, such as members of the genus Listeria. In these embodiments,the hydrophilic agents may include a combination of nutrients andselective inhibitors that favor the growth of Listeria species overother microorganisms that may be present in a surface sample. Forexample, the reagent strip may include a pancreatic digest of casein, aproteose peptone such as proteose peptone #3, yeast extract, a bufferingsystem such as disodium phosphate and monopotassium phosphate, sodiumchloride, lithium chloride, naladixic acid, acriflavin, moxalactam, andpolymyxin B sulfate in concentrations that are effective to support thegrowth of Listeria species and/or inhibit the growth non-Listeriaspecies. The reagent strip may further include an indicator system, suchas a chromogenic enzyme substrate (e.g.,6-Chloro-3-indoxyl-α-D-mannopyranoside) and, optionally, a correspondingenzyme inducer (e.g., 1-O-methyl-α-D-mannopyranoside) at effectiveconcentrations to detect the presence of Listeria species in the sample.

Microbial Detection or Enumeration Articles

The present invention includes articles used to detect and/or enumeratemicroorganisms in a surface sample. Such articles, which include thepreviously-described sample housings, comprise various combinations ofelements (e.g., cover sheet, sample collector, barrier layer, bottommember) described above.

FIG. 6 illustrates one embodiment of an article 600 for samplecollection and microbial enumeration. The article 600 is comprised ofsample collector 610 and reagent strip 680 elements, constructed asdescribed above. The sample collector 610 comprises a water-impervioussubstrate 612 and a bonded material 624. The bonded material 624 isattached to the lower major surface of substrate 612 by an adhesive 622.In this embodiment, the reagent strip 680 comprises a self-supportingsubstrate 682 with upper and lower surfaces, at least a part of onemajor surface coated with a hydrophilic agent 686. Also shown in FIG. 6is the optional adhesive 684. The sample collector 610 and reagent strip680 are attached in this embodiment by a double-sided adhesive tape 640at the hinge region 645. Certain low-adhesion adhesive mixtures, such asthose described in U.S. Pat. No. 5,118,750, may be used on at least oneside of the double-sided adhesive tape 640 to form a detachable (andre-attachable) attachment between the sample collector 610 and reagentstrip 680.

Preferably, the sample collector 610 and reagent strip 680 are attachednear one edge of the article 600, forming a hinge region 645. A skilledperson will recognize other suitable ways to form the hinge region 645,such as adhesives, heat-bonding, ultrasonic welding, and the like. It isalso contemplated that the sample collector 610 and reagent strip 680may be formed from a single piece of water-impervious material, with ahinge region 645 formed by folding the material back on itself (notshown).

FIG. 7 illustrates an alternative embodiment of an article 700 forsample collection and microbial enumeration. The article 700 iscomprised of sample collector 710, cover sheet 730, and bottom member750 elements, the elements constructed as described above. The coversheet 730 is comprised of a substrate 732, an adhesive layer 734, andpowder 736. The sample collector 710 can be attached to the cover sheet730 via a double-sided adhesive tape 740. The sample collector 710includes a bonded material 724, for sample collection, attached to thesubstrate 712 by an adhesive layer 722. In this embodiment, the samplecollector 710 includes a perforation 726 near the hinge region 745, foreasy removal of the sample collector 710 from the article 700. Thesample collector also includes a tab 728 to grasp the sample collector710 during handling. A bottom member 750 is attached to the samplecollector 710 via a double-sided adhesive tape 740. The bottom member750 comprises a substrate 752 and an optional attachment structure 758,to which the sample collector 710 can be affixed. The attachmentstructure 758 may comprise an adhesive, a component of a hook-and-loopattachment means, or the like, which can be used to hold a samplecollector 710 in place. In certain embodiments, the sample collector 710major surface that faces the attachment structure 758 may comprise arelease material, to control adhesion of the sample collector 710 to theattachment structure 758, when said attachment structure 758 comprisesan adhesive. Release coatings are known in the art and are routinelyused to reduce the adhesion between adhesives and plastic films.

FIG. 8 illustrates an alternative embodiment of an article 800 forsample collection and microbial enumeration. The article 800 iscomprised of cover sheet 830, barrier layer 870, sample collector 810,and bottom member 850 elements, the elements constructed as describedabove. The cover sheet 830 is comprised of a substrate 832, an adhesivelayer 834, and powder 836. The sample collector 810 can be attached tothe cover sheet 830 via a double-sided adhesive tape 840. The samplecollector 810 includes a bonded material 824, for sample collection,attached to the substrate 812 by an adhesive layer 822. In thisembodiment, the sample collector 810 includes a perforation 826 near thehinge region 845, for easy removal of the sample collector 810 from thearticle 800. The sample collector also includes a tab 828 to grasp thesample collector 810 during handling. A barrier layer 870 is attached tothe sample collector 810 via a double-sided adhesive tape 840. Thebarrier layer 870 also includes a perforation 872 near the hinge region845 and a tab 874 to grasp the barrier layer 870 during handling. Abottom member 850 is attached to the barrier layer 870 via adouble-sided adhesive tape 840. The bottom member 850 comprises asubstrate 852 and an optional attachment structure 858, to which thesample collector 810 can be affixed. The attachment structure 858 maycomprise an adhesive, a component of a hook-and-loop attachment means,or the like, which can be used to hold a sample collector 810 in place.In certain embodiments, the barrier layer 870 major surface that facesthe attachment structure 858 may comprise a release material, to controladhesion of the sample collector 810 to the attachment structure 858,when said attachment structure 858 comprises an adhesive. Releasecoatings are known in the art and are routinely used to reduce theadhesion between adhesives and plastic films.

Samples and Microorganisms

One aspect of the present invention is that it may be used to detectorganisms present on a wide variety of surfaces. The devices and methodsof the present invention may be used for a variety of applications whereit is desirable to test the organisms present on a surface, including,but not limited to, food surfaces (e.g. beef carcasses, exteriorsurfaces of produce), food processing surfaces, water or water filmsurfaces, patient surfaces, patient treatment surfaces, hospitalenvironmental surfaces, clinic environmental surfaces, and forensicenvironmental surfaces. The samples may consist substantially of solid,semi-solid, gelatinous, or liquid material, alone or in variouscombinations. The apparatus and system of the invention, as well as theinventive methods, may be used to determine, qualitatively orquantitatively, the presence of one or more microorganisms of interest.

An exemplary clinical analyte of interest to detect is Staphylococcusaureus (“S. aureus”). This is a pathogen causing a wide spectrum ofinfections including: superficial lesions such as small skin abscessesand wound infections; systemic and life threatening conditions such asendocarditis, pneumonia and septicemia; as well as toxinoses such asfood poisoning and toxic shock syndrome. Some strains (e.g.,Methicillin-Resistant S. aureus or MRSA) are resistant to all but a fewselect antibiotics.

Exemplary analytes of interest to detect in food processing areas aremembers of the genus Listeria. Listeria are classified as gram-positive,rod-shaped bacteria and consist of the species Listeria monocytogenes,L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, and L. grayi.Among these, L. monocytogenes is responsible for the majority of humanlisteriosis cases and immunocompromised, pregnant women, elderly, andnewborns have increased susceptibility to infection. The most commonsymptoms of listeriosis are septicemia, meningitis, and miscarriages.

Other microorganisms of particular interest for analytical purposesinclude prokaryotic and eukaryotic organisms, particularly Gram positivebacteria, Gram negative bacteria, fungi, mycoplasma, and yeast.Particularly relevant organisms include members of the familyEnterobacteriaceae, or the family Micrococcaceae or the generaStaphylococcus spp., Streptococcus spp., Pseudomonas spp., Enterococcusspp., Salmonella spp., Legionella spp., Shigella spp. Yersinia spp.,Enterobacter spp., Escherichia spp., Bacillus spp., Vibrio spp.,Clostridium spp., Corynebacteria spp. as well as, Aspergillus spp.,Fusarium spp., and Candida spp. Particularly virulent organisms includeStaphylococcus aureus (including resistant strains such as MethicillinResistant Staphylococcus aureus (MRSA)), S. epidermidis, Streptococcuspneumoniae, S. agalactiae, S. pyogenes, Enterococcus faecalis,Vancomycin Resistant Enterococcus (VRE), Vancomycin ResistantStaphylococcus aureus (VRSA), Vancomycin Intermediate-resistantStaphylococcus aureus (VISA), Bacillus anthracis, Pseudomonasaeruginosa, Escherichia coli, Aspergillus niger, A. fumigatus, A.clavatus, Fusarium solani, F. oxysporum, F. chlamydosporum, Vibriocholera, V. parahemolyticus, Salmonella cholerasuis, S. typhi, S.typhimurium, Candida albicans, C. glabrata, C. krusei, Enterobactersakazakii, Escherichia coli O157, ESBL-containing microorganisms, andmultiple drug resistant Gram negative rods (MDR).

Articles of Manufacture

Sample housings and/or articles of the invention can be combined withpackaging material and sold as a kit for sampling and detectingmicroorganisms on surfaces. For example, the kits may comprise two ormore components (e.g., bottom member, spacer, barrier layer, cover sheetand/or reagent strip; each component as described above) packagedtogether. In some embodiments, two or more of the components (e.g.bottom member, barrier layer, and cover sheet) may be provided attachedto each other, preferably forming a hinge region, as shown in theillustrated embodiments. In these embodiments, certain components (e.g.,the sample collector, reagent strip, and/or the barrier layer) may bedetachable. In other embodiments, the components may be providedseparately and can be assembled during use. The kits may furthercomprise sampling and testing accessories, such as a sample suspendingmedium, a pipette, a label, a sample carrier, or a glove.

Environmental Sampling and Detection Methods

The present disclosure includes methods for detecting microorganisms onan environmental surface. The methods may be used to detect the presenceor absence of a target microorganism by observing an indicator ofmicrobial growth of the target microorganism. The methods further may beused to enumerate microorganisms in a sample. The elements that are usedin these methods (e.g. sample collector, cover sheet, barrier layer,etc.) may be provided separately or may be parts of an assembly, such asthe sample housings described above. Furthermore, the individualelements and/or assemblies may be provided in a kit, as described above.The embodiments described herein

In one embodiment, the method comprises providing a liquid samplesuspending medium; a sample collector such as sample collector 210 (FIG.2B), a sample housing such as sample housing 460 (FIG. 4) comprising abottom member 450 and cover sheet 430, and a reagent strip such asreagent strip 580 (FIG. 5). The method further comprises contacting oneof the major surfaces, such as a bonded material 224, of the samplecollector 210 with the surface or material to be tested. Typically, theoperator may use sterile forceps, or the like, or may use a gloved handto hold the sample collector 210 while the sample is collected. Thesample collector 210 is brought into physical contact (e.g., touching,wiping, or rubbing) with a prescribed surface area, such as 5 cm by 5cm, 10 cm by 10 cm or 25 cm by 25 cm, in order to estimate the number oforganisms in an area of known dimensions. In this embodiment, thebarrier layer 470 is lifted away from the bottom member 450 and thesample collector 210 is placed in contact with the attachment structure458 with the sample oriented toward the barrier layer 470. The methodfurther comprises applying the sample suspending medium to the bondedmaterial 224 of the sample collector 210 comprising the sample. Thesample suspending medium may be applied using a pipette or by othersuitable means. After adding the sample suspending medium, the barrierlayer 470 may be lowered. Optionally, the sample may be incubated for aperiod of time, such as at least 30 minutes, at least 60 minutes, or atleast 120 minutes, depending upon the target microorganism, to allow therecovery of injured microorganisms. The incubation temperature may bechosen according to the target microorganism. After the optionalincubation period, the cover sheet 430 is lifted and the barrier layer470 is removed, for example by separating the barrier layer 470 at theperforation 472. The method further comprises contacting the lowersurface of the cover sheet 430 with the sample suspending medium to forma hydrated gel. This may be accomplished, for example, by grasping thecover sheet 430 with one hand, grasping the bottom member 450 with theother hand, and lifting the cover sheet 430. Although the hydrated gelforms within seconds, it is preferable to allow the gel to form for atleast 5-10 minutes, more preferably, at least 10-30 minutes beforeopening the device to expose the hydrated gel. The gel may remainattached to the cover sheet 430, the gel may remain attached to thesample collector 210, or separate portions of the gel may remainattached to the cover sheet 430 and to the sample collector 210.Optionally, the method further comprises placing a coated surface of thereagent strip 580 in contact with the hydrated gel. While the coversheet 430 is raised, a reagent strip 580 may be inserted into the samplehousing 460, with the hydrophilic agent 586 of the reagent strip 580oriented toward the gel. The sample housing 460 is incubated for aperiod of time to allow the number of microorganisms present in thesample to increase and/or to allow the metabolic activity of themicroorganisms to cause a detectable change in an indicator. The deviceis typically incubated at temperatures from 25-45° C., preferably 25-37°C. In certain embodiments, the microorganisms form discrete colonies inthe hydrated gel. The number of microorganisms in the original samplemay be enumerated by counting colonies.

In another embodiment, the method comprises providing a liquid samplesuspending medium; any sample collector (see, for example, FIGS. 1.2A-B); a sample housing such as sample housing 360 (FIG. 3A) comprisinga bottom member 350, a barrier layer 370, and a cover sheet 330; and areagent strip such as the reagent strip 580 (FIG. 5). The method furthercomprises contacting one of the major surfaces of the substrate orbonded material of the sample collector with the surface or material tobe tested, as described above. The sample collector can be placed intothe sample housing 360 with the sample oriented toward the barrier layer370. In this embodiment, the barrier layer 370 can be lifted and thesample collector can be placed into the well formed by aperture 357 withthe sample oriented upward (toward the barrier layer 370). In someembodiments, the sample collector may be sized and shaped so that itfits completely inside the well formed by aperture 357. The methodfurther comprises dispensing the sample suspending medium into the wellcontaining the sample collector. The sample suspending medium may bedispensed using a pipette or by other suitable means. After adding thesample suspending medium, the barrier layer 370 may be lowered.Optionally, the sample may be incubated for a period of time, such as atleast 30 minutes, at least 60 minutes, or at least 120 minutes,depending upon the target microorganism, to allow the recovery ofinjured microorganisms. After the optional incubation period, the coversheet 330 is lifted and the barrier layer 370 is removed, for example byseparating the barrier layer 370 at the perforation 372. The methodfurther comprises contacting the lower surface of the cover sheet 330with the sample suspending medium to form a hydrated gel, as describedabove. Although the hydrated gel forms within seconds, it is preferableto allow the gel to form preferably for at least 5-10 minutes or morepreferably for at least 10-30 minutes before opening the device toexpose the hydrated gel. Optionally, the method further comprisesplacing a coated surface of the reagent strip 580 in contact with thehydrated gel. In this process, the cover sheet 330 is lifted to exposethe hydrated gel, the reagent strip 580 is positioned in and/or near theaperture 357 in the sample housing 360, and the cover sheet 330 islowered to bring the reagent strip into contact with the hydrated gel.The sample housing 360 is then incubated for a period of time, afterwhich the device is observed for an indicator of microbial growth. Thedevice is typically incubated at temperatures from 20-45° C., preferably25-37° C. The device is incubated for a period of time (e.g., 18-48hours, depending on the growth rate of the target microorganisms and theindicator system) to allow the number of microorganisms present in thesample to increase and/or to allow the metabolic activity of themicroorganisms to cause a detectable change in an indicator. In certainembodiments, the microorganisms form discrete colonies in the hydratedgel. The number of microorganisms in the original sample may beenumerated by counting colonies.

EXAMPLES Example 1 Detection and Enumeration of Listeria innocua from aSurface

Preparation of Reagent Strips

Liquid growth media for the growth and detection of Listeria species wasmade according to the formula listed in Table 1. The enzyme inducer(1-O-methyl-α-D-mannopyranoside) and chromogenic enzyme substrate(6-Chloro-3-indoxyl-α-D-mannopyranoside) were obtained from BiosynthInternational (Naperville, Ill.). M150 Ethanol-washed Guar was obtainedfrom Danisco (Kreutzlingen, Switzerland).

TABLE 1 Broth medium for growth and detection of Listeria species.Ingredient Concentration (g/L) Pancreatic Digest of Casein 10.0 ProteosePeptone #3 10.0 Nalidixic Acid 0.036 Acriflavin 0.03 Moxalactam 0.03Polymyxin B Sulfate (7690 units/mg) 0.001 Yeast Extract 8.0 SodiumChloride 40.0 Lithium Chloride 4.5 Disodium Phosphate 12.0 Monopotassiumphosphate 6.0 1-O-methyl-α-D-mannopyranoside 5.06-Chloro-3-indoxyl-α-D-mannopyranoside 2.0 Xanthan gum 8.0 M150Ethanol-washed Guar 4.0

The substrate used for coating the reagent strip was 8.5″ wide, 2.91 mil(0.07 mm) thick clear polyester film. The liquid media was coated on thefirst side of the substrate using a knife coater with a 7 mil (0.18 mm)gap and subsequently passing the coated substrate through a drying ovenset at approximately 230° F. (110° C.). The oven drying time wasapproximately two and half minutes. The coating and drying procedure wasrepeated on the opposite side of the substrate, resulting in a plasticfilm that was coated on both sides with the same growth medium. Theapproximate coating weight for each side of the coated film was 0.230g/24 in² (0.230 g/155 cm²). The double-coated substrate was die cut intocircles, each circle having a diameter of 2.875 in. (7.30 cm).

Preparation of the Sample Housing Devices

Sample housing devices were constructed by assembling the followingcomponents into a single unit: a bottom member, a foam spacer, a barrierlayer, and a cover sheet. The foam spacer was attached to the bottommember by an adhesive that was coated onto the bottom member, asdescribed below. The barrier layer was attached along one edge of thefoam spacer using ⅜ in. (9.5 mm)-wide double-sided adhesive tape. Thecover film was attached (along the same edge) to the barrier layer usingthe double-sided adhesive tape. Drawings of this construction can beseen in FIGS. 3A and 3B.

The bottom member consisted of 6.3 mil (0.16 mm) thick polycoated paper(83 pound HD RHI-Lease FA 34 Yellow Grid, Grade 406-83010, obtained fromWausau-Moisinee Paper Corp., Rhinelander, Wis.). A yellow grid(perpendicular lines spaced 1 cm apart over the entire area) was printedonto the non-silicone-treated (“bottom”) side of the paper. Thesilicone-treated (“top”) side of the paper was coated with an iso-octylacrylate/acrylamide copolymer adhesive (96 wt. % iso-octyl acrylate and4 wt. % acrylamide obtained from 3M Company, St. Paul, Minn.; coatingweight approximately 145-200 mg/200 cm²).

The spacer consisted of polystyrene foam material (CL3V Capliner, White,8.5 inches (21.6 cm) wide, 20 mil (0.51 mm) thick; obtained fromAmerican Fuji Seal, Bardstown, Ky.). A 2.875 inch (7.30 cm) circle wasdie cut and removed from the spacer material. The spacer material wasthen laminated to the adhesive coated side of the bottom member byrunning the bottom member and die-cut spacer through a nip roller. Thecircle cut out of the spacer material formed a circular depression or“well” and the bottom member formed the bottom surface of the well inthe laminate, as shown in FIG. 3A.

The barrier film consisted of polyester (200 gauge polyester film, ColorK399 Light Blue, obtained from CPFilms Inc., Martinsville, Va.). Arelease was coated on one side of the barrier film. The barrier film wasattached along one edge of the foam spacer using double-sided adhesivetape such that the release coated side of the barrier film was facingthe bottom member.

The cover sheet was constructed from a 1.6 mil (0.04 mm) thickbiaxially-orientated polypropylene (BOPP) film that was previouslycoated with an iso-octyl acrylate/acrylamide copolymer adhesive (96 wt.% iso-octyl acrylate and 4 wt. % acrylamide, coating weightapproximately 165-260 mg/200 cm²). The adhesive coated side of the BOPPfilm was subsequently coated with M150 non-ethanol washed guar gumpowder (obtained from Danisco; powder coating weight approximately0.30-0.60 g/24 in² (0.30-0.60 g/155 cm²). The cover sheet was attachedto the barrier layer with double sided adhesive tape, as describedabove.

Preparation of Sample Collectors

Two types of sample collectors were constructed. Type I samplecollectors were constructed using clear 3 mil polyester film. The filmwas die cut into 2.875 inch (7.3 cm) diameter circles. Type II samplecollectors were constructed by laminating nonwoven materials to aplastic substrate. For type II sample collectors, a tackified highpressure sensitive iso-octyl acrylate/acrylic acid copolymer adhesive(96 wt. % iso-octyl acrylate and 4 wt. % acrylic acid, Part NumberAZ-1229, 3M Company, St. Paul, Minn., coating thickness wasapproximately 2 mils (0.05 mm)) was laminated to a 5 mil (0.13 mm) thickwhite polyester film (226Melinex, obtained from DuPont Teijin Films,Hopewell, Va.) and an absorbent material (shown in Table 2) wassubsequently laminated onto the adhesive. After the laminates were made,the type II sample collectors were die cut into 2.875 inch (7.24 cm)diameter circles. Table 2 lists the sample collectors used in theseexperiments.

TABLE 2 Sample Collectors Sample Collector Description Type Nonwoven A 3mil (0.08 mm) I None clear polyester film B Polyester II Knitted Loop(Style No. WW1112, laminate Gehring Textiles, Inc., Garden City, NY) CPolyester II 40 HEX VisPore Film (0.000125 laminate inch caliper,Tredegar Film Products, Richmond, VA)Preparation of Inoculated Surfaces

Listeria innocua (ATCC#33091) was grown in tryptic soy broth with 0.6%yeast extract overnight at 35° C. Seventy-five microliters of theovernight culture was diluted into 50 mL of tryptic soy broth with 0.6%yeast extract. The suspension was shaken and two 0.5 mL samples werespread over 4×4 inch sections of flat, sterile stainless steel using asterile Dacron polyester tipped applicator. The stainless steel surfaceswere allowed to dry at room temperature. After the steel surfaces weredry, the devices described above were used to collect and quantify themicrobes on the surface of the stainless steel.

Surface Testing Procedure

The experimental procedure described below was evaluated in comparisonto a standard method, which consisted of i) a sponge premoistened withneutralizing buffer (Nasco, 18-oz. Whirl-Pak Hydrated Speci-Sponge Bag,Product ID—BO1422WA, from Hardy Diagnostics, Santa Maria, Calif.) wasrubbed over a 4 inch by 4 inch (103 cm²) inoculated area and placed intothe bag from which it came, ii) five milliliters of buffered peptonewater was added to the bag containing the sponge and the bag wasmanually massaged for approximately 30 seconds to release the bacteriafrom the sponge, iii) the bag containing the sponge and buffered peptonewater was allowed to stand at room temperature for 1-1.5 hours, iv)three milliliters of the liquid suspension was removed from the bagusing a pipette and was dispensed into a Petrifilm™ EnvironmentalListeria plate (3M Company, St. Paul, Minn.), and v) the Petrifilm platewas incubated and the appearance of any bacterial colonies wasinterpreted according to the manufacturer's instructions.

In the experimental procedure, the sample collector was rubbed over the4 inch by 4 inch (103 cm²) section of the inoculated, dried stainlesssteel surface for a period of 30 to 60 seconds. In some instances, thesample collector was dry. In other instances, the sample collector waspremoistened by applying a light aerosol (approximately 0.5-1.0milliliters of buffered peptone water) to the sample collector using aspray bottle).

The sample collector was placed into the sample housing in the “well”formed by the foam spacer, with the sample-side facing upward (towardthe polyester barrier layer). The sample housing was subsequently heldat room temperature for approximately 25 minutes. The cover film andbarrier film were lifted off the foam spacer, the barrier film wasremoved, and 3 mLs of buffered peptone water was pipetted onto thesample collector. The cover film was reattached to the foam spacer withdouble sided adhesive tape and the cover film was subsequently loweredonto the foam spacer, bringing the powdered guar gum into contact withthe buffered peptone water to form a gel. The sample housing was kept ona level surface for approximately 60 minutes to allow for some growth ofthe microorganisms. Afterward, the cover film was lifted and thedouble-coated reagent strip was inserted into the sample housing suchthat one side of the reagent strip contacted the sample collector andthe other side contacted the hydrated portion of the cover film. Thesample housing was then placed into an incubator at 35° C. for 26 hours.The reagent strip was examined for the presence of bacterial colonies(which typically appeared as small dots of various sizes, having varyingshades of reddish color). The results of this test are shown in Table 3and Table 4.

TABLE 3 Microbial Counts (Each number represents a colony count from asingle plate.) Sample Collector Colony Forming Units/Sample Material DrySample Collector Wet Sample Collector Standard Method NA 29 PolyesterFilm 0 70 Knitted Loop 35 32 40 HEX VisPore Film 17 41

TABLE 4 Colony Appearance Sample Collector Colony Appearance after 26Hrs. of Incubation Material Dry Sample Collector Wet Sample CollectorPolyester No visible colonies. Colonies were the same Film - 3 mil size,shape, and color (red) as the standard method. Knitted Loop Colonieswere Colonies were the same approximately the same size size, shape, andcolor as and shape as the standard the standard method. method, althoughthe colony color was a lighter shade of red and the colony margins wereless distinct than the colonies observed in the standard method. 40 HEXColonies were Colonies were VisPore Film approximately the same sizeapproximately the same and shape as the standard size and shape as themethod, although the colony standard method, color was a lighter shadeof although the colony color red and the colony margins was a lightershade of red were less distinct than the and the colony margins coloniesobserved in the were less distinct than the standard method. coloniesobserved in the standard method.

Example 2 Detection and Enumeration of Staphylococcus Aureus from aSurface

Preparation of the Sample Housing Devices

The sample housing devices used in this experiment were 3M PETRIFILMStaph Express (STX) Count System plates obtained from 3M Company (St.Paul, Minn.). The dry media in each plate was hydrated with 1 milliliterof Butterfield's phosphate diluent and was allowed to gel prior to usingthe plates in this experiment.

Preparation of Sample Collectors

Type I sample collectors were constructed using clear 3 mil polyesterfilm as described in Example 1. Type II sample collectors wereconstructed by laminating the designated bonded material (e.g.,cheesecloth) to clear 3 mil polyethylene film using a pressure-sensitiveacrylic-based adhesive. CEREX G192988 nonwoven material was obtainedfrom CEREX Advanced Fabrics (Cantonment, Fla.). Hanes Wetlaid Hydroguard150 HEM PET/cellulose was obtained from Hanes Industries (Conover,N.C.). The Hanes material was pretreated with SPAN 20 (obtained fromUniquema, Stanford, Fla.) to make it hydrophilic. The pretreatmentconsisted of wiping the fabric material with solution of SPAN-20 (2.5%w/v SPAN-20-sorbitan monolaurate in 97.5% w/v isopropyl alcohol) untilthe fabric was saturated with the solution. The fabric was allowed toair dry at room temperature. A portion of the treated fabric was testedby transferring a few drops of water onto a surface of the fabric toobserve that the water was absorbed (wicked) into the fabric.

Preparation of Inoculated Surfaces

Staphylococcus aureus ATCC 25923 was grown in tryptic soy broth for 18hours at 35° C. Twenty microliters of the overnight culture was dilutedinto 99 mL of Butterfield's phosphate diluent and the resulting mixturewas shaken to uniformly distribute the bacteria in suspension. Onemilliliter of the buffered bacterial suspension was transferred to asecond bottle (99 mL) of Butterfield's phosphate diluent. The dilutedsuspension was shaken and three 0.5 mL samples were spread over 4×4 inchsections of separate flat, sterile stainless steel coupons using asterile glass spreader. The stainless steel surfaces were allowed to dryat room temperature for 30-60 minutes in a laminar flow hood. It wasestimated that each stainless steel coupon was inoculated withapproximately 60 colony-forming units (CFU) of S. aureus cells in thisprocedure. After the steel surfaces are dry, the sample collectorsdescribed above were used to collect and quantify the microbes on thesurface of the stainless steel.

Surface Testing Procedure

The sample collectors were moistened by applying a light aerosol ofButterfield's phosphate diluent (approximately 0.5-1.0 mL) to the samplecollector using a spray bottle. Samples were collected by vigorouslyrubbing a surface of the sample collector over a 4 inch by 4 inch (103cm²) section of the inoculated, dried stainless steel surface. Thesurface was rubbed side-to-side in one direction, and then rubbedside-to-side in a direction that was perpendicular to the direction inwhich the surface was initially rubbed.

The cover sheet of a prehydrated PETRIFILM STX plate was lifted,exposing the well in the center of the foam spacer (i.e., the gel formedby the dehydrated media remained attached to the cover sheet). Thesample collector was placed into the sample housing (in the well formedby the aperture in the foam spacer) with the sample-side facing upward(toward the cover sheet). The cover sheet was lowered, contacting thehydrated gel with the sample on the sample collector. The sample housingwas placed into an incubator at 35° C. for about 18 hours and thecolonies, which appeared as small red dots, were counted. The number ofcolonies in each plate is shown in Table 5. The absence of colonyforming units in the experiment with a polyethylene film sample as thecollector may be due to the loss of viability of the of bacteriainoculated onto the test surfaces during the course of the experiment.

TABLE 5 Microbial Counts (Each number represents a colony count from asingle plate.) Sample Collector Base Material/ Colony Forming BondedMaterial Units/Sample Polyethylene Film/ 0 None Polyethylene Film/ 10Cheesecloth Polyethylene Film/ 8 CEREX G192988

The present invention has now been described with reference to severalspecific embodiments foreseen by the inventor for which enablingdescriptions are available. Insubstantial modifications of theinvention, including modifications not presently foreseen, maynonetheless constitute equivalents thereto. Thus, the scope of thepresent invention should not be limited by the details and structuresdescribed herein, but rather solely by the following claims, andequivalents thereto.

What is claimed is:
 1. A method for detecting microorganisms on anenvironmental surface, the method comprising: providing; a barrierlayer; a sample-suspending medium; a sample collector comprising asubstrate with upper and lower major surfaces wherein at least one majorsurface is water-impervious; a sample housing comprising: a bottommember comprising a self-supporting substrate with upper and lower majorsurfaces; a cover sheet comprising a substrate with upper and lowermajor surfaces wherein at least part of one major surface is coated witha cold water soluble powder including at least one gelling agent; and areagent strip comprising a self-supporting substrate with upper andlower major surfaces wherein at least part of one major surface iscoated with a cold water soluble gelling agent and a hydrophilic agentselected from the group consisting of a nutrient for growingmicroorganisms, a selective agent, an indicator, and combinations of anytwo or more of the foregoing; obtaining a sample on at least one majorsurface of the sample collector; forming an assembly by placing thesample-collector into the sample housing with the at least one majorsurface comprising the sample oriented toward the cover sheet; applyingthe sample-suspending medium to the sample collector major surfacecomprising the sample; inserting the water-resistant barrier layerbetween the sample collector and the cover sheet, thereby preventing thesample-suspending medium from contacting at least a part of the coversheet; incubating the assembly for a period of time; removing at least aportion of the barrier layer from the sample housing; contacting thelower major surface of the cover sheet with the sample-suspending mediumto form a hydrated gel; placing a coated surface of the reagent strip incontact with the hydrated gel; incubating the sample housing for aperiod of time; and observing an indicator of microbial growth.
 2. Themethod according to claim 1, the method further comprising the step ofenumerating microorganisms in the sample.
 3. The method according toclaim 1, wherein observing an indicator of microbial growth comprisesobserving an indicator of growth of a microorganism selected from agroup consisting of Listeria spp., Salmonella spp., Enterobacteriaceae,Clostridium spp., Enterococcus spp., Yersinia spp. Bacillus spp.,Escherichia coli, Staphylococcus spp., and Extended-Spectrumβ-Lactamase-containing microorganisms.